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Shen Y, Thng DKH, Wong ALA, Toh TB. Mechanistic insights and the clinical prospects of targeted therapies for glioblastoma: a comprehensive review. Exp Hematol Oncol 2024; 13:40. [PMID: 38615034 PMCID: PMC11015656 DOI: 10.1186/s40164-024-00512-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2023] [Accepted: 04/08/2024] [Indexed: 04/15/2024] Open
Abstract
Glioblastoma (GBM) is a fatal brain tumour that is traditionally diagnosed based on histological features. Recent molecular profiling studies have reshaped the World Health Organization approach in the classification of central nervous system tumours to include more pathogenetic hallmarks. These studies have revealed that multiple oncogenic pathways are dysregulated, which contributes to the aggressiveness and resistance of GBM. Such findings have shed light on the molecular vulnerability of GBM and have shifted the disease management paradigm from chemotherapy to targeted therapies. Targeted drugs have been developed to inhibit oncogenic targets in GBM, including receptors involved in the angiogenic axis, the signal transducer and activator of transcription 3 (STAT3), the PI3K/AKT/mTOR signalling pathway, the ubiquitination-proteasome pathway, as well as IDH1/2 pathway. While certain targeted drugs showed promising results in vivo, the translatability of such preclinical achievements in GBM remains a barrier. We also discuss the recent developments and clinical assessments of targeted drugs, as well as the prospects of cell-based therapies and combinatorial therapy as novel ways to target GBM. Targeted treatments have demonstrated preclinical efficacy over chemotherapy as an alternative or adjuvant to the current standard of care for GBM, but their clinical efficacy remains hindered by challenges such as blood-brain barrier penetrance of the drugs. The development of combinatorial targeted therapies is expected to improve therapeutic efficacy and overcome drug resistance.
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Affiliation(s)
- Yating Shen
- The N.1 Institute for Health (N.1), National University of Singapore, Singapore, Singapore
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
| | - Dexter Kai Hao Thng
- The N.1 Institute for Health (N.1), National University of Singapore, Singapore, Singapore
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
| | - Andrea Li Ann Wong
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
- Department of Haematology-Oncology, National University Hospital, Singapore, Singapore
| | - Tan Boon Toh
- The N.1 Institute for Health (N.1), National University of Singapore, Singapore, Singapore.
- The Institute for Digital Medicine (WisDM), National University of Singapore, Singapore, Singapore.
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2
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Ninomiya I, Koyama A, Otsu Y, Onodera O, Kanazawa M. Regeneration of the cerebral cortex by direct chemical reprogramming of macrophages into neuronal cells in acute ischemic stroke. Front Cell Neurosci 2023; 17:1225504. [PMID: 37636590 PMCID: PMC10457112 DOI: 10.3389/fncel.2023.1225504] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2023] [Accepted: 07/31/2023] [Indexed: 08/29/2023] Open
Abstract
Theoretically, direct chemical reprogramming of somatic cells into neurons in the infarct area represents a promising regenerative therapy for ischemic stroke. Previous studies have reported that human fibroblasts and astrocytes transdifferentiate into neuronal cells in the presence of small molecules without introducing ectopic transgenes. However, the optimal combination of small molecules for the transdifferentiation of macrophages into neurons has not yet been determined. The authors hypothesized that a combination of small molecules could induce the transdifferentiation of monocyte-derived macrophages into neurons and that the administration of this combination may be a regenerative therapy for ischemic stroke because monocytes and macrophages are directly involved in the ischemic area. Transcriptomes and morphologies of the cells were compared before and after stimulation using RNA sequencing and immunofluorescence staining. Microscopic analyses were also performed to identify cell markers and evaluate functional recovery by blinded examination following the administration of small molecules after ischemic stroke in CB-17 mice. In this study, an essential combination of six small molecules [CHIR99021, Dorsomorphin, Forskolin, isoxazole-9 (ISX-9), Y27632, and DB2313] that transdifferentiated monocyte-derived macrophages into neurons in vitro was identified. Moreover, administration of six small molecules after cerebral ischemia in model animals generated a new neuronal layer in the infarct cortex by converting macrophages into neuronal cells, ultimately improving neurological function. These results suggest that altering the transdifferentiation of monocyte-derived macrophages by the small molecules to adjust their adaptive response will facilitate the development of regenerative therapies for ischemic stroke.
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Affiliation(s)
- Itaru Ninomiya
- Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan
| | - Akihide Koyama
- Department of Legal Medicine, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
| | - Yutaka Otsu
- Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan
| | - Osamu Onodera
- Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan
| | - Masato Kanazawa
- Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan
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3
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Heat shock response enhanced by cell culture treatment in mouse embryonic stem cell-derived proliferating neural stem cells. PLoS One 2021; 16:e0249954. [PMID: 33852623 PMCID: PMC8046196 DOI: 10.1371/journal.pone.0249954] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2020] [Accepted: 03/27/2021] [Indexed: 12/16/2022] Open
Abstract
Cells have a regulatory mechanism known as heat shock (HS) response, which induces the expression of HS genes and proteins in response to heat and other cellular stresses. Exposure to moderate HS results in beneficial effects, such as thermotolerance and promotes survival, whereas excessive HS causes cell death. The effect of HS on cells depends on both exogenous factors, including the temperature and duration of heat application, and endogenous factors, such as the degree of cell differentiation. Neural stem cells (NSCs) can self-renew and differentiate into neurons and glial cells, but the changes in the HS response of symmetrically proliferating NSCs in culture are unclear. We evaluated the HS response of homogeneous proliferating NSCs derived from mouse embryonic stem cells during the proliferative phase and its effect on survival and cell death in vitro. The number of adherent cells and the expression ratios of HS protein (Hsp)40 and Hsp70 genes after exposure to HS for 20 min at temperatures above 43°C significantly increased with the extension of the culture period before exposure to HS. In contrast, caspase activity was significantly decreased by extension of the culture period before exposure to HS and suppressed the decrease in cell viability. These results suggest that the culture period before HS remarkably affects the HS response, influencing the expression of HS genes and cell survival of proliferating NSCs in culture.
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Reprogramming of chimpanzee fibroblasts into a multipotent cancerous but not fully pluripotent state by transducing iPSC factors in 2i/LIF culture. Differentiation 2020; 112:67-76. [DOI: 10.1016/j.diff.2020.01.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2019] [Revised: 10/23/2019] [Accepted: 01/06/2020] [Indexed: 02/07/2023]
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Pouya A, Rassouli H, Rezaei-Larijani M, Salekdeh GH, Baharvand H. SOX2 protein transduction directly converts human fibroblasts into oligodendrocyte-like cells. Biochem Biophys Res Commun 2020; 525:S0006-291X(20)30315-6. [PMID: 32070492 DOI: 10.1016/j.bbrc.2020.02.047] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Accepted: 02/07/2020] [Indexed: 01/26/2023]
Abstract
Oligodendrocyte precursor cells (OPCs) are ideal therapeutic cells for treatment of spinal cord injuries and diseases that affect myelin. However, it is necessary to generate a cell population with a low risk of teratoma formation and oncogenesis from a patient's somatic cells. In this study, we investigated the direct reprogramming of fibroblasts to oligodendrocyte-like cells in one step with a safe non-genetic delivery method that used protein transduction. Cell morphology and the lineage-specific marker expression profile indicated that human foreskin fibroblasts (HFFs) were converted into oligodendrocyte-like cells by the application of pluripotency factors and the use of a permissible induction medium. Our data demonstrated that SOX2 was sufficient to directly drive OPC fate conversion from HFF by a genetic-free approach. Therefore, this work has provided a strategy to OPC reprogramming by a non-integrating approach for future use in disease modeling and may ultimately provide applications for patient-specific cell-based regenerative medicine.
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Affiliation(s)
- Alireza Pouya
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hassan Rassouli
- Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Medical Laser, Medical Laser Research Center, Yara Institute, ACECR, Tehran, Iran
| | - Mehran Rezaei-Larijani
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Ghasem Hosseini Salekdeh
- Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, Tehran, Iran.
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6
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HORISAWA K, SUZUKI A. Direct cell-fate conversion of somatic cells: Toward regenerative medicine and industries. PROCEEDINGS OF THE JAPAN ACADEMY. SERIES B, PHYSICAL AND BIOLOGICAL SCIENCES 2020; 96:131-158. [PMID: 32281550 PMCID: PMC7247973 DOI: 10.2183/pjab.96.012] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
Cells of multicellular organisms have diverse characteristics despite having the same genetic identity. The distinctive phenotype of each cell is determined by molecular mechanisms such as epigenetic changes that occur throughout the lifetime of an individual. Recently, technologies that enable modification of the fate of somatic cells have been developed, and the number of studies using these technologies has increased drastically in the last decade. Various cell types, including neuronal cells, cardiomyocytes, and hepatocytes, have been generated using these technologies. Although most direct reprogramming methods employ forced transduction of a defined sets of transcription factors to reprogram cells in a manner similar to induced pluripotent cell technology, many other strategies, such as methods utilizing chemical compounds and microRNAs to change the fate of somatic cells, have also been developed. In this review, we summarize transcription factor-based reprogramming and various other reprogramming methods. Additionally, we describe the various industrial applications of direct reprogramming technologies.
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Affiliation(s)
- Kenichi HORISAWA
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
| | - Atsushi SUZUKI
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
- Correspondence should be addressed: A. Suzuki, Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (e-mail: )
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7
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Erharter A, Rizzi S, Mertens J, Edenhofer F. Take the shortcut - direct conversion of somatic cells into induced neural stem cells and their biomedical applications. FEBS Lett 2019; 593:3353-3369. [PMID: 31663609 PMCID: PMC6916337 DOI: 10.1002/1873-3468.13656] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2019] [Revised: 10/12/2019] [Accepted: 10/14/2019] [Indexed: 12/20/2022]
Abstract
Second-generation reprogramming of somatic cells directly into the cell type of interest avoids induction of pluripotency and subsequent cumbersome differentiation procedures. Several recent studies have reported direct conversion of human somatic cells into stably proliferating induced neural stem cells (iNSCs). Importantly, iNSCs are easier, faster, and more cost-efficient to generate than induced pluripotent stem cells (iPSCs), and also have a higher level of clinical safety. Stably, self-renewing iNSCs can be derived from different cellular sources, such as skin fibroblasts and peripheral blood mononuclear cells, and readily differentiate into neuronal and glial lineages that are indistinguishable from their iPSC-derived counterparts or from NSCs isolated from primary tissues. This review focuses on the derivation and characterization of iNSCs and their biomedical applications. We first outline different approaches to generate iNSCs and then discuss the underlying molecular mechanisms. Finally, we summarize the preclinical validation of iNSCs to highlight that these cells are promising targets for disease modeling, autologous cell therapy, and precision medicine.
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Affiliation(s)
- Anita Erharter
- Department of Molecular Biology & CMBIGenomics, Stem Cell Biology & Regenerative MedicineLeopold‐Franzens‐University InnsbruckAustria
| | - Sandra Rizzi
- Department of Molecular Biology & CMBIGenomics, Stem Cell Biology & Regenerative MedicineLeopold‐Franzens‐University InnsbruckAustria
- Institute of PharmacologyMedical University InnsbruckAustria
| | - Jerome Mertens
- Department of Molecular Biology & CMBIGenomics, Stem Cell Biology & Regenerative MedicineLeopold‐Franzens‐University InnsbruckAustria
| | - Frank Edenhofer
- Department of Molecular Biology & CMBIGenomics, Stem Cell Biology & Regenerative MedicineLeopold‐Franzens‐University InnsbruckAustria
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Toyoshima M, Jiang X, Ogawa T, Ohnishi T, Yoshihara S, Balan S, Yoshikawa T, Hirokawa N. Enhanced carbonyl stress induces irreversible multimerization of CRMP2 in schizophrenia pathogenesis. Life Sci Alliance 2019; 2:2/5/e201900478. [PMID: 31591136 PMCID: PMC6781483 DOI: 10.26508/lsa.201900478] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2019] [Revised: 09/17/2019] [Accepted: 09/19/2019] [Indexed: 12/31/2022] Open
Abstract
Enhanced carbonyl stress results in neurodevelopmental deficits by affecting microtubule function through the formation of irreversible dysfunctional multimer of carbonylated CRMP2. Enhanced carbonyl stress underlies a subset of schizophrenia, but its causal effects remain elusive. Here, we elucidated the molecular mechanism underlying the effects of carbonyl stress in iPS cells in which the gene encoding zinc metalloenzyme glyoxalase I (GLO1), a crucial enzyme for the clearance of carbonyl stress, was disrupted. The iPS cells exhibited significant cellular and developmental deficits, and hyper-carbonylation of collapsing response mediator protein 2 (CRMP2). Structural and biochemical analyses revealed an array of multiple carbonylation sites in the functional motifs of CRMP2, particularly D-hook (for dimerization) and T-site (for tetramerization), which are critical for the activity of the CRMP2 tetramer. Interestingly, carbonylated CRMP2 was stacked in the multimer conformation by irreversible cross-linking, resulting in loss of its unique function to bundle microtubules. Thus, the present study revealed that the enhanced carbonyl stress stemmed from the genetic aberrations results in neurodevelopmental deficits through the formation of irreversible dysfunctional multimer of carbonylated CRMP2.
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Affiliation(s)
- Manabu Toyoshima
- Laboratory for Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Japan
| | - Xuguang Jiang
- Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
| | - Tadayuki Ogawa
- Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
| | - Tetsuo Ohnishi
- Laboratory for Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Japan
| | - Shogo Yoshihara
- Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
| | - Shabeesh Balan
- Laboratory for Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Japan
| | - Takeo Yoshikawa
- Laboratory for Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Japan
| | - Nobutaka Hirokawa
- Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Tokyo, Japan .,Center of Excellence in Genome Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia
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9
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Inoue Y, Kishida T, Kotani SI, Akiyoshi M, Taga H, Seki M, Ukimura O, Mazda O. Direct conversion of fibroblasts into urothelial cells that may be recruited to regenerating mucosa of injured urinary bladder. Sci Rep 2019; 9:13850. [PMID: 31554870 PMCID: PMC6761134 DOI: 10.1038/s41598-019-50388-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2019] [Accepted: 09/11/2019] [Indexed: 02/08/2023] Open
Abstract
Urothelial cells play essential roles in protection of urine exudation and bacterial invasion at the urothelial mucosa, so that defect or damage of urothelial cells associated with urinary tract diseases may cause serious problems. If a sufficient number of functional urothelial cells are prepared in culture and transplanted into the damaged urothelial lesions, such technology may provide beneficial effects to patients with diseases of the urinary tract. Here we found that human adult dermal fibroblasts were converted into urothelial cells by transducing genes for four transcription factors, FOXA1, TP63, MYCL and KLF4 (FTLK). The directly converted urothelial cells (dUCs) formed cobblestone-like colonies and expressed urothelium-specific markers. dUCs were successfully expanded and enriched after serial passages using a specific medium that we optimized for the cells. The passaged dUCs showed similar genome-wide gene expression profiles to normal urothelial cells and had a barrier function. The FTLK-transduced fibroblasts were also converted into urothelial cells in vivo and recruited to the regenerating urothelial tissue after they were transplanted into the bladder of mice with interstitial cystitis. Our technology may provide a promising solution for a number of patients with urinary tract disorders.
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Affiliation(s)
- Yuta Inoue
- Department of Immunology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan.,Department of Urology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan
| | - Tsunao Kishida
- Department of Immunology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan
| | - Shin-Ichiro Kotani
- Department of Immunology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan
| | - Mika Akiyoshi
- Department of Immunology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan.,CellAxia Inc. 1-10-9-6F Nihonbashi Horidome-cho, Chuo-ku, Tokyo, 103-0012, Japan
| | - Hideto Taga
- Department of Immunology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan.,Department of Urology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan
| | - Makoto Seki
- Department of Immunology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan.,CellAxia Inc. 1-10-9-6F Nihonbashi Horidome-cho, Chuo-ku, Tokyo, 103-0012, Japan
| | - Osamu Ukimura
- Department of Urology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan
| | - Osam Mazda
- Department of Immunology, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan.
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Flitsch LJ, Brüstle O. Evolving principles underlying neural lineage conversion and their relevance for biomedical translation. F1000Res 2019; 8. [PMID: 31559012 PMCID: PMC6743253 DOI: 10.12688/f1000research.18926.1] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 08/23/2019] [Indexed: 12/19/2022] Open
Abstract
Scientific and technological advances of the past decade have shed light on the mechanisms underlying cell fate acquisition, including its transcriptional and epigenetic regulation during embryonic development. This knowledge has enabled us to purposefully engineer cell fates
in vitro by manipulating expression levels of lineage-instructing transcription factors. Here, we review the state of the art in the cell programming field with a focus on the derivation of neural cells. We reflect on what we know about the mechanisms underlying fate changes in general and on the degree of epigenetic remodeling conveyed by the distinct reprogramming and direct conversion strategies available. Moreover, we discuss the implications of residual epigenetic memory for biomedical applications such as disease modeling and neuroregeneration. Finally, we cover recent developments approaching cell fate conversion in the living brain and define questions which need to be addressed before cell programming can become an integral part of translational medicine.
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Affiliation(s)
- Lea Jessica Flitsch
- Institute of Reconstructive Neurobiology, University of Bonn School of Medicine & University Hospital Bonn, Bonn, North Rhine Wesphalia, 53127, Germany
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, University of Bonn School of Medicine & University Hospital Bonn, Bonn, North Rhine Wesphalia, 53127, Germany
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Khoshchehreh R, Totonchi M, Carlos Ramirez J, Torres R, Baharvand H, Aicher A, Ebrahimi M, Heeschen C. Epigenetic reprogramming of primary pancreatic cancer cells counteracts their in vivo tumourigenicity. Oncogene 2019; 38:6226-6239. [PMID: 31308488 PMCID: PMC6756074 DOI: 10.1038/s41388-019-0871-x] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2019] [Revised: 05/03/2019] [Accepted: 05/03/2019] [Indexed: 12/22/2022]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) arises through accumulation of multiple genetic alterations. However, cancer cells also acquire and depend on cancer-specific epigenetic changes. To conclusively demonstrate the crucial relevance of the epigenetic programme for the tumourigenicity of the cancer cells, we used cellular reprogramming technology to reverse these epigenetic changes. We reprogrammed human PDAC cultures using three different techniques - (1) lentivirally via induction of Yamanaka Factors (OSKM), (2) the pluripotency-associated gene OCT4 and the microRNA mir-302, or (3) using episomal vectors as a safer alternative without genomic integration. We found that induction with episomal vectors was the most efficient method to reprogram primary human PDAC cultures as well as primary human fibroblasts that served as positive controls. Successful reprogramming was evidenced by immunostaining, alkaline phosphatase staining, and real-time PCR. Intriguingly, reprogramming of primary human PDAC cultures drastically reduced their in vivo tumourigenicity, which appeared to be driven by the cells' enhanced differentiation and loss of stemness upon transplantation. Our study demonstrates that reprogrammed primary PDAC cultures are functionally distinct from parental PDAC cells resulting in drastically reduced tumourigenicity in vitro and in vivo. Thus, epigenetic alterations account at least in part for the tumourigenicity and aggressiveness of pancreatic cancer, supporting the notion that epigenetic modulators could be a suitable approach to improve the dismal outcome of patients with pancreatic cancer.
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Affiliation(s)
- Reyhaneh Khoshchehreh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Mehdi Totonchi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
- Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
| | | | - Raul Torres
- Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, 28029, Spain
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, 08036, Spain
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Alexandra Aicher
- Gene and Stem Cell Therapy Program, Centenary Institute, the University of Sydney, Camperdown, 2050, NSW, Australia.
- Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, 28029, Madrid, Spain.
| | - Marzieh Ebrahimi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
- Department of Developmental Biology, University of Science and Culture, Tehran, Iran.
| | - Christopher Heeschen
- Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, 28029, Madrid, Spain.
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Nishihara K, Shiga T, Nakamura E, Akiyama T, Sasaki T, Suzuki S, Ko MSH, Tada N, Okano H, Akamatsu W. Induced Pluripotent Stem Cells Reprogrammed with Three Inhibitors Show Accelerated Differentiation Potentials with High Levels of 2-Cell Stage Marker Expression. Stem Cell Reports 2019; 12:305-318. [PMID: 30713040 PMCID: PMC6373546 DOI: 10.1016/j.stemcr.2018.12.018] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2017] [Revised: 12/29/2018] [Accepted: 12/30/2018] [Indexed: 02/06/2023] Open
Abstract
Although pluripotent stem cells can generate various types of differentiated cells, it is unclear why lineage-committed stem/progenitor cells derived from pluripotent stem cells are decelerated and why the differentiation-resistant propensity of embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC)-derived cells is predominant compared with the in vivo equivalents derived from embryonic/adult tissues. In this study, we demonstrated that iPSCs reprogrammed and maintained with three chemical inhibitors of the fibroblast growth factor 4-mitogen-activated protein kinase cascade and GSK3β (3i) could be differentiated into all three germ layers more efficiently than the iPSCs reprogrammed without the 3i chemicals, even though they were maintained with 3i chemicals once they were reprogrammed. Although the iPSCs reprogrammed with 3i had increased numbers of Zscan4-positive cells, the Zscan4-positive cells among iPSCs that were reprogrammed without 3i did not have an accelerated differentiation ability. These observations suggest that 3i exposure during the reprogramming period determines the accelerated differentiation/maturation potentials of iPSCs that are stably maintained at the distinct state.
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Affiliation(s)
- Koji Nishihara
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Takahiro Shiga
- Center for Genomic and Regenerative Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Eri Nakamura
- Research Institute for Diseases of Old Age, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Tomohiko Akiyama
- Department of Systems Medicine, Sakaguchi Laboratory, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Takashi Sasaki
- Center for Supercentenarian Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Sadafumi Suzuki
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Minoru S H Ko
- Department of Systems Medicine, Sakaguchi Laboratory, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Norihiro Tada
- Research Institute for Diseases of Old Age, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
| | - Wado Akamatsu
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Center for Genomic and Regenerative Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
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13
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Ang CE, Ma Q, Wapinski OL, Fan S, Flynn RA, Lee QY, Coe B, Onoguchi M, Olmos VH, Do BT, Dukes-Rimsky L, Xu J, Tanabe K, Wang L, Elling U, Penninger JM, Zhao Y, Qu K, Eichler EE, Srivastava A, Wernig M, Chang HY. The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders. eLife 2019; 8:41770. [PMID: 30628890 PMCID: PMC6380841 DOI: 10.7554/elife.41770] [Citation(s) in RCA: 54] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2018] [Accepted: 01/07/2019] [Indexed: 12/25/2022] Open
Abstract
Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.
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Affiliation(s)
- Cheen Euong Ang
- Department of Pathology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, United States.,Department of Bioengineering, Stanford University, Stanford, United States
| | - Qing Ma
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States.,Department of Dermatology, Stanford University, Stanford, United States.,Department of Genetics, Stanford University, Stanford, United States
| | - Orly L Wapinski
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States.,Department of Dermatology, Stanford University, Stanford, United States.,Department of Genetics, Stanford University, Stanford, United States
| | - ShengHua Fan
- JC Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, United States
| | - Ryan A Flynn
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States.,Department of Dermatology, Stanford University, Stanford, United States.,Department of Genetics, Stanford University, Stanford, United States
| | - Qian Yi Lee
- Department of Pathology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, United States.,Department of Bioengineering, Stanford University, Stanford, United States
| | - Bradley Coe
- Department of Genome Sciences, Howard Hughes Medical Institute, University of Washington, Seattle, United States
| | - Masahiro Onoguchi
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States.,Department of Dermatology, Stanford University, Stanford, United States.,Department of Genetics, Stanford University, Stanford, United States
| | - Victor Hipolito Olmos
- Department of Pathology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, United States
| | - Brian T Do
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States
| | - Lynn Dukes-Rimsky
- JC Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, United States
| | - Jin Xu
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States
| | - Koji Tanabe
- Department of Pathology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, United States
| | - LiangJiang Wang
- Department of Genetics and Biochemistry, Clemson University, Clemson, United States
| | - Ulrich Elling
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter, Vienna, Austria
| | - Josef M Penninger
- Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter, Vienna, Austria
| | - Yang Zhao
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States
| | - Kun Qu
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States.,Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter, Vienna, Austria
| | - Evan E Eichler
- Department of Genome Sciences, Howard Hughes Medical Institute, University of Washington, Seattle, United States
| | - Anand Srivastava
- JC Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, United States.,Department of Genetics and Biochemistry, Clemson University, Clemson, United States
| | - Marius Wernig
- Department of Pathology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, United States
| | - Howard Y Chang
- Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States
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14
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Saito S, Lin YC, Nakamura Y, Eckner R, Wuputra K, Kuo KK, Lin CS, Yokoyama KK. Potential application of cell reprogramming techniques for cancer research. Cell Mol Life Sci 2019; 76:45-65. [PMID: 30283976 PMCID: PMC6326983 DOI: 10.1007/s00018-018-2924-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2018] [Revised: 09/15/2018] [Accepted: 09/19/2018] [Indexed: 02/07/2023]
Abstract
The ability to control the transition from an undifferentiated stem cell to a specific cell fate is one of the key techniques that are required for the application of interventional technologies to regenerative medicine and the treatment of tumors and metastases and of neurodegenerative diseases. Reprogramming technologies, which include somatic cell nuclear transfer, induced pluripotent stem cells, and the direct reprogramming of specific cell lineages, have the potential to alter cell plasticity in translational medicine for cancer treatment. The characterization of cancer stem cells (CSCs), the identification of oncogene and tumor suppressor genes for CSCs, and the epigenetic study of CSCs and their microenvironments are important topics. This review summarizes the application of cell reprogramming technologies to cancer modeling and treatment and discusses possible obstacles, such as genetic and epigenetic alterations in cancer cells, as well as the strategies that can be used to overcome these obstacles to cancer research.
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Affiliation(s)
- Shigeo Saito
- Saito Laboratory of Cell Technology, Yaita, Tochigi, 329-1571, Japan
- College of Engineering, Nihon University, Koriyama, Fukushima, 963-8642, Japan
| | - Ying-Chu Lin
- School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung, 807, Taiwan
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, 305-0074, Japan
| | - Richard Eckner
- Department of Biochemistry and Molecular Biology, Rutgers, New Jersey Medical School-Rutgers, The State University of New Jersey, Newark, NJ, 07101, USA
| | - Kenly Wuputra
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, 807, Taiwan
| | - Kung-Kai Kuo
- Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, 807, Taiwan
| | - Chang-Shen Lin
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, 807, Taiwan.
- Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, 804, Taiwan.
| | - Kazunari K Yokoyama
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, 807, Taiwan.
- Faculty of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-0033, Japan.
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15
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McCaughey-Chapman A, Connor B. Human Cortical Neuron Generation Using Cell Reprogramming: A Review of Recent Advances. Stem Cells Dev 2018; 27:1674-1692. [DOI: 10.1089/scd.2018.0122] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Affiliation(s)
- Amy McCaughey-Chapman
- Department of Pharmacology and Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
| | - Bronwen Connor
- Department of Pharmacology and Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
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16
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Tsuji O, Sugai K, Yamaguchi R, Tashiro S, Nagoshi N, Kohyama J, Iida T, Ohkubo T, Itakura G, Isoda M, Shinozaki M, Fujiyoshi K, Kanemura Y, Yamanaka S, Nakamura M, Okano H. Concise Review: Laying the Groundwork for a First-In-Human Study of an Induced Pluripotent Stem Cell-Based Intervention for Spinal Cord Injury. Stem Cells 2018; 37:6-13. [PMID: 30371964 PMCID: PMC7379555 DOI: 10.1002/stem.2926] [Citation(s) in RCA: 89] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Revised: 08/16/2018] [Accepted: 09/22/2018] [Indexed: 12/21/2022]
Abstract
There have been numerous attempts to develop stem cell transplantation approaches to promote the regeneration of spinal cord injury (SCI). Our multicenter team is currently planning to launch a first-in-human clinical study of an induced pluripotent stem cell (iPSC)-based cell transplant intervention for subacute SCI. This trial was conducted as class I regenerative medicine protocol as provided for under Japan's Act on the Safety of Regenerative Medicine, using neural stem/progenitor cells derived from a clinical-grade, integration-free human "iPSC stock" generated by the Kyoto University Center for iPS Cell Research and Application. In the present article, we describe how we are preparing to initiate this clinical study, including addressing the issues of safety and tumorigenesis as well as practical problems that must be overcome to enable the development of therapeutic interventions for patients with chronic SCI. Stem Cells 2019;37:6-13.
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Affiliation(s)
- Osahiko Tsuji
- Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan
| | - Keiko Sugai
- Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan
| | - Ryo Yamaguchi
- Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan.,Department of Physiology, Keio University School of Medicine, Tokyo, Japan.,Regenerative & Cellular Medicine Office, Sumitomo Dainippon Pharma Co., Ltd., Kobe, Japan
| | - Syoichi Tashiro
- Department of Rehabilitation Medicine, Keio University School of Medicine, Tokyo, Japan
| | - Narihito Nagoshi
- Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan
| | - Jun Kohyama
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Tsuyoshi Iida
- Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan.,Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Toshiki Ohkubo
- Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan.,Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Go Itakura
- Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan.,Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Miho Isoda
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan.,Regenerative & Cellular Medicine Office, Sumitomo Dainippon Pharma Co., Ltd., Kobe, Japan
| | - Munehisa Shinozaki
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Kanehiro Fujiyoshi
- Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan.,Department of Orthopaedic Surgery, National Hospital Organization Murayama Medical Center, Tokyo, Japan
| | - Yonehiro Kanemura
- Department of Biomedical Research and Innovation, Institute for Clinical Research and Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka, Japan
| | - Shinya Yamanaka
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Masaya Nakamura
- Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
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17
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Connor B, Firmin E, McCaughey-Chapman A, Monk R, Lee K, Liot S, Geiger J, Rudolph C, Jones K. Conversion of adult human fibroblasts into neural precursor cells using chemically modified mRNA. Heliyon 2018; 4:e00918. [PMID: 30450440 PMCID: PMC6226601 DOI: 10.1016/j.heliyon.2018.e00918] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2018] [Revised: 10/11/2018] [Accepted: 11/02/2018] [Indexed: 12/14/2022] Open
Abstract
Direct reprogramming offers a unique approach by which to generate neural lineages for the study and treatment of neurological disorders. Our objective is to develop a clinically viable reprogramming strategy to generate neural precursor cells for the treatment of neurological disorders through cell replacement therapy. We initially developed a method for directly generating neural precursor cells (iNPs) from adult human fibroblasts by transient expression of the neural transcription factors, SOX2 and PAX6 using plasmid DNA. This study advances these findings by examining the use of chemically modified mRNA (cmRNA) for direct-to-iNP reprogramming. Chemically modified mRNA has the benefit of being extremely stable and non-immunogenic, offering a clinically suitable gene delivery system. The use of SOX2 and PAX6 cmRNA resulted in high co-transfection efficiency and cell viability compared with plasmid transfection. Neural positioning and fate determinant genes were observed throughout reprogramming with ion channel and synaptic marker genes detected during differentiation. Differentiation of cmRNA-derived iNPs generated immature GABAergic or glutamatergic neuronal phenotypes in conjunction with astrocytes. This represents the first time a cmRNA approach has been used to directly reprogram adult human fibroblasts to iNPs, potentially providing an efficient system by which to generate human neurons for both research and clinical application.
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Affiliation(s)
- Bronwen Connor
- Department of Pharmacology & Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
| | - Erin Firmin
- Department of Pharmacology & Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
| | - Amy McCaughey-Chapman
- Department of Pharmacology & Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
| | - Ruth Monk
- Department of Pharmacology & Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
| | - Kevin Lee
- Department of Physiology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
| | - Sophie Liot
- Department of Pharmacology & Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
| | | | | | - Kathryn Jones
- Department of Pharmacology & Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
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18
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Directing neuronal cell fate in vitro : Achievements and challenges. Prog Neurobiol 2018; 168:42-68. [DOI: 10.1016/j.pneurobio.2018.04.003] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2017] [Revised: 03/30/2018] [Accepted: 04/05/2018] [Indexed: 12/22/2022]
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19
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Golas MM. Human cellular models of medium spiny neuron development and Huntington disease. Life Sci 2018; 209:179-196. [PMID: 30031060 DOI: 10.1016/j.lfs.2018.07.030] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2018] [Revised: 06/22/2018] [Accepted: 07/17/2018] [Indexed: 12/24/2022]
Abstract
The loss of gamma-aminobutyric acid (GABA)-ergic medium spiny neurons (MSNs) in the striatum is the hallmark of Huntington disease (HD), an incurable neurodegenerative disorder characterized by progressive motor, psychiatric, and cognitive symptoms. Transplantation of MSNs or their precursors represents a promising treatment strategy for HD. In initial clinical trials in which HD patients received fetal neurografts directly into the striatum without a pretransplant cell-differentiation step, some patients exhibited temporary benefits. Meanwhile, major challenges related to graft overgrowth, insufficient survival of grafted cells, and limited availability of donated fetal tissue remain. Thus, the development of approaches that allow modeling of MSN differentiation and HD development in cell culture platforms may improve our understanding of HD and translate, ultimately, into HD treatment options. Here, recent advances in the in vitro differentiation of MSNs derived from fetal neural stem cells/progenitor cells (NSCs/NPCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and induced NSCs (iNSCs) as well as advances in direct transdifferentiation are reviewed. Progress in non-allele specific and allele specific gene editing of HTT is presented as well. Cell characterization approaches involving phenotyping as well as in vitro and in vivo functional assays are also discussed.
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Affiliation(s)
- Monika M Golas
- Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 3, Building 1233, DK-8000 Aarhus C, Denmark; Department of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
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20
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Okolie O, Irvin DM, Bago JR, Sheets K, Satterlee A, Carey-Ewend AG, Lettry V, Dumitru R, Elton S, Ewend MG, Miller CR, Hingtgen SD. Intra-cavity stem cell therapy inhibits tumor progression in a novel murine model of medulloblastoma surgical resection. PLoS One 2018; 13:e0198596. [PMID: 29990322 PMCID: PMC6038981 DOI: 10.1371/journal.pone.0198596] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2017] [Accepted: 05/22/2018] [Indexed: 12/02/2022] Open
Abstract
Background Cytotoxic neural stem cells (NSCs) have emerged as a promising treatment for Medulloblastoma (MB), the most common malignant primary pediatric brain tumor. The lack of accurate pre-clinical models incorporating surgical resection and tumor recurrence limits advancement in post-surgical MB treatments. Using cell lines from two of the 5 distinct MB molecular sub-groups, in this study, we developed an image-guided mouse model of MB surgical resection and investigate intra-cavity NSC therapy for post-operative MB. Methods Using D283 and Daoy human MB cells engineered to express multi-modality optical reporters, we created the first image-guided resection model of orthotopic MB. Brain-derived NSCs and novel induced NSCs (iNSCs) generated from pediatric skin were engineered to express the pro-drug/enzyme therapy thymidine kinase/ganciclovir, seeded into the post-operative cavity, and used to investigate intra-cavity therapy for post-surgical MB. Results We found that surgery reduced MB volumes by 92%, and the rate of post-operative MB regrowth increased 3-fold compared to pre-resection growth. Real-time imaging showed NSCs rapidly homed to MB, migrating 1.6-fold faster and 2-fold farther in the presence of tumors, and co-localized with MB present in the contra-lateral hemisphere. Seeding of cytotoxic NSCs into the post-operative surgical cavity decreased MB volumes 15-fold and extended median survival 133%. As an initial step towards novel autologous therapy in human MB patients, we found skin-derived iNSCs homed to MB cells, while intra-cavity iNSC therapy suppressed post-surgical tumor growth and prolonged survival of MB-bearing mice by 123%. Conclusions We report a novel image-guided model of MB resection/recurrence and provide new evidence of cytotoxic NSCs/iNSCs delivered into the surgical cavity effectively target residual MB foci.
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Affiliation(s)
- Onyinyechukwu Okolie
- Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - David M. Irvin
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Division of Neuropathology, Department of Pathology and Laboratory Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Department of Neurology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Neuroscience Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Juli R. Bago
- Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Kevin Sheets
- Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Andrew Satterlee
- Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Abigail G. Carey-Ewend
- Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Vivien Lettry
- Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Raluca Dumitru
- UNC Human Pluripotent Stem Cell Core, Genetics Department, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Scott Elton
- Department of Neurosurgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Matthew G. Ewend
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Department of Neurosurgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - C. Ryan Miller
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Division of Neuropathology, Department of Pathology and Laboratory Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Department of Neurology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Neuroscience Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- UNC Neuroscience Center, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Shawn D. Hingtgen
- Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Department of Neurosurgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- UNC Neuroscience Center, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- * E-mail:
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21
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Loera-Valencia R, Piras A, Ismail MAM, Manchanda S, Eyjolfsdottir H, Saido TC, Johansson J, Eriksdotter M, Winblad B, Nilsson P. Targeting Alzheimer's disease with gene and cell therapies. J Intern Med 2018; 284:2-36. [PMID: 29582495 DOI: 10.1111/joim.12759] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Alzheimer's disease (AD) causes dementia in both young and old people affecting more than 40 million people worldwide. The two neuropathological hallmarks of the disease, amyloid beta (Aβ) plaques and neurofibrillary tangles consisting of protein tau are considered the major contributors to the disease. However, a more complete picture reveals significant neurodegeneration and decreased cell survival, neuroinflammation, changes in protein and energy homeostasis and alterations in lipid and cholesterol metabolism. In addition, gene and cell therapies for severe neurodegenerative disorders have recently improved technically in terms of safety and efficiency and have translated to the clinic showing encouraging results. Here, we review broadly current data within the field for potential targets that could modify AD through gene and cell therapy strategies. We envision that not only Aβ will be targeted in a disease-modifying treatment strategy but rather that a combination of treatments, possibly at different intervention times may prove beneficial in curing this devastating disease. These include decreased tau pathology, neuronal growth factors to support neurons and modulation of neuroinflammation for an appropriate immune response. Furthermore, cell based therapies may represent potential strategies in the future.
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Affiliation(s)
- R Loera-Valencia
- Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, Solna, Sweden
| | - A Piras
- Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, Solna, Sweden
| | - M A M Ismail
- Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, Solna, Sweden.,Theme Neuro, Diseases of the Nervous System Patient Flow, Karolinska University Hospital, Huddinge, Sweden
| | - S Manchanda
- Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, Solna, Sweden
| | - H Eyjolfsdottir
- Division of Clinical Geriatrics, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Huddinge, Sweden.,Theme Aging, Karolinska University Hospital, Huddinge, Sweden
| | - T C Saido
- RIKEN Brain Science Institute, Wako, Saitama, Japan
| | - J Johansson
- Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, Solna, Sweden
| | - M Eriksdotter
- Division of Clinical Geriatrics, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Huddinge, Sweden.,Theme Aging, Karolinska University Hospital, Huddinge, Sweden
| | - B Winblad
- Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, Solna, Sweden.,Theme Aging, Karolinska University Hospital, Huddinge, Sweden
| | - P Nilsson
- Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, Solna, Sweden
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22
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Transdifferentiation of human adult peripheral blood T cells into neurons. Proc Natl Acad Sci U S A 2018; 115:6470-6475. [PMID: 29866841 DOI: 10.1073/pnas.1720273115] [Citation(s) in RCA: 66] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Human cell models for disease based on induced pluripotent stem (iPS) cells have proven to be powerful new assets for investigating disease mechanisms. New insights have been obtained studying single mutations using isogenic controls generated by gene targeting. Modeling complex, multigenetic traits using patient-derived iPS cells is much more challenging due to line-to-line variability and technical limitations of scaling to dozens or more patients. Induced neuronal (iN) cells reprogrammed directly from dermal fibroblasts or urinary epithelia could be obtained from many donors, but such donor cells are heterogeneous, show interindividual variability, and must be extensively expanded, which can introduce random mutations. Moreover, derivation of dermal fibroblasts requires invasive biopsies. Here we show that human adult peripheral blood mononuclear cells, as well as defined purified T lymphocytes, can be directly converted into fully functional iN cells, demonstrating that terminally differentiated human cells can be efficiently transdifferentiated into a distantly related lineage. T cell-derived iN cells, generated by nonintegrating gene delivery, showed stereotypical neuronal morphologies and expressed multiple pan-neuronal markers, fired action potentials, and were able to form functional synapses. These cells were stable in the absence of exogenous reprogramming factors. Small molecule addition and optimized culture systems have yielded conversion efficiencies of up to 6.2%, resulting in the generation of >50,000 iN cells from 1 mL of peripheral blood in a single step without the need for initial expansion. Thus, our method allows the generation of sufficient neurons for experimental interrogation from a defined, homogeneous, and readily accessible donor cell population.
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23
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Omrani MR, Yaqubi M, Mohammadnia A. Transcription Factors in Regulatory and Protein Subnetworks during Generation of Neural Stem Cells and Neurons from Direct Reprogramming of Non-fibroblastic Cell Sources. Neuroscience 2018; 380:63-77. [PMID: 29653196 DOI: 10.1016/j.neuroscience.2018.03.033] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2017] [Revised: 03/19/2018] [Accepted: 03/20/2018] [Indexed: 12/31/2022]
Abstract
Direct reprogramming of non-fibroblastic cells to the neuronal cell types including induced neurons (iNs) and induced neural stem cells (iNSCs) has provided an alternative approach for the direct reprogramming of fibroblasts to those cells. However, to increase the efficiency of the reprogramming process the underlying mechanisms should be clarified. In the current study, we analyzed the gene expression profiles of five different cellular conversions to understand the most significant molecular mechanisms and transcription factors (TFs) underlying each conversion. For each conversion, we found the list of differentially expressed genes (DEGs) and the list of differentially expressed TFs (DE-TFs) which regulate expression of DEGs. Moreover, we constructed gene regulatory networks based on the TF-binding sites' data and found the most central regulators and the most active part of the networks. Furthermore, protein complexes were identified from constructed protein-protein interaction networks for DE-TFs. Finally, we proposed a list of main regulators for each conversion; for example, in the direct conversion of epithelial-like cells (ECs) to iNSCs, combination of centrality with active modules or protein complex analyses highlighted the role of POU3F2, BACH1, AR, PBX1, SOX2 and NANOG genes in this conversion. To the best of our knowledge, this study is the first one that analyzed the direct conversion of non-fibroblastic cells toward iNs and iNSCs and we believe that the expression manipulation of identified genes may increase efficiency of these processes.
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Affiliation(s)
- Mohammad Reza Omrani
- National Institute of Genetics Engineering and Biotechnology (NIGEB), Tehran, Iran
| | - Moein Yaqubi
- Department of Psychiatry, Ludmer Centre for Neuroinformatics and Mental Health, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada.
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Park HS, Kwon H, Yu J, Bae Y, Park JY, Choi KA, Choi Y, Hong S. Precise nanoinjection delivery of plasmid DNA into a single fibroblast for direct conversion of astrocyte. ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2018; 46:1114-1122. [PMID: 29506416 DOI: 10.1080/21691401.2018.1446019] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
Direct conversion is a powerful approach to safely generate mature neural lineages with potential for treatment of neurological disorders. Astrocytes play a crucial role in neuronal homeostasis and their dysfunctions contribute to several neurodegenerative diseases. Using a single-cell approach for precision, we describe here a robust method using optimized DNA amounts for the direct conversion of mouse fibroblasts to astrocytes. Controlled amount of the reprogramming factors Oct4, Sox2, Klf4 and cMyc was directly delivered into a single fibroblast cell. Consequently, 2500 DNA molecules, no more or less, were found to be the optimal amount that dramatically increased the expression levels of the astrocyte-specific markers GFAP and S100b and the demethylation gene TET1, the expression of which was sustained to maintain astrocyte functionality. The converted astrocytes showed glutamate uptake ability and electrophysiological activity. Furthermore, we demonstrated a potential mechanism whereby fibroblast was directly converted into astrocyte at a single-cell level; this was achieved by activating BMP2 pathway through direct binding of Sox2 protein to BMP2 gene. This study suggests that nanotechnology for directly injecting plasmid DNAs into cell nuclei may help understand such a conversion at single-cell level.
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Affiliation(s)
- Hang-Soo Park
- a Department of Integrated Biomedical and Life Science , Korea University , Seoul , Republic of Korea
| | - Hyosung Kwon
- b Department of Bio-convergence Engineering , Korea University , Seoul , Republic of Korea
| | - Jewon Yu
- b Department of Bio-convergence Engineering , Korea University , Seoul , Republic of Korea
| | - Yeonju Bae
- a Department of Integrated Biomedical and Life Science , Korea University , Seoul , Republic of Korea
| | - Jae-Yong Park
- a Department of Integrated Biomedical and Life Science , Korea University , Seoul , Republic of Korea.,c School of Biosystem and Biomedical Science , Korea University , Seoul , Republic of Korea
| | - Kyung-Ah Choi
- c School of Biosystem and Biomedical Science , Korea University , Seoul , Republic of Korea
| | - Yeonho Choi
- b Department of Bio-convergence Engineering , Korea University , Seoul , Republic of Korea.,d School of Biomedical Engineering , Korea University , Seoul , Republic of Korea
| | - Sunghoi Hong
- a Department of Integrated Biomedical and Life Science , Korea University , Seoul , Republic of Korea.,c School of Biosystem and Biomedical Science , Korea University , Seoul , Republic of Korea
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25
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Shahbazi E, Mirakhori F, Ezzatizadeh V, Baharvand H. Reprogramming of somatic cells to induced neural stem cells. Methods 2018; 133:21-28. [PMID: 28939501 DOI: 10.1016/j.ymeth.2017.09.007] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2017] [Revised: 09/02/2017] [Accepted: 09/12/2017] [Indexed: 12/26/2022] Open
Abstract
Recent investigations have demonstrated that defined sets of exogenous factors (chemical and/or biochemical) can convert human and mouse somatic cells into induced neural stem cells (iNSCs). Considering the self-renewal and multi-potential differentiation capabilities of iNSCs, generation of these cells has considerably enhanced cell therapy for treatment of neurodegenerative disorders. These cells can also serve as models for investigation of the mechanism(s) underlying neurodegenerative diseases and as an asset in drug discovery. Meanwhile, using the process of direct conversion/transdifferentiation, by bypassing pluripotent state and consequently reducing tumorigenesis and genetic instability risks, establishment of several desired cells are feasible. In this review, we describe the pros and cons of different methods employed to directly reprogram somatic cells to iNSCs along with the progress of iNSCs applications and the future challenges.
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Affiliation(s)
- Ebrahim Shahbazi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Fahimeh Mirakhori
- Institute for Cell Engineering, Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Vahid Ezzatizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, Tehran, Iran.
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26
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Bagó JR, Okolie O, Dumitru R, Ewend MG, Parker JS, Werff RV, Underhill TM, Schmid RS, Miller CR, Hingtgen SD. Tumor-homing cytotoxic human induced neural stem cells for cancer therapy. Sci Transl Med 2018; 9:9/375/eaah6510. [PMID: 28148846 DOI: 10.1126/scitranslmed.aah6510] [Citation(s) in RCA: 71] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2015] [Revised: 07/26/2016] [Accepted: 10/05/2016] [Indexed: 12/13/2022]
Abstract
Engineered neural stem cells (NSCs) are a promising approach to treating glioblastoma (GBM). The ideal NSC drug carrier for clinical use should be easily isolated and autologous to avoid immune rejection. We transdifferentiated (TD) human fibroblasts into tumor-homing early-stage induced NSCs (h-iNSCTE), engineered them to express optical reporters and different therapeutic gene products, and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSCTE in patient-derived GBM models of surgical and nonsurgical disease. Molecular and functional analysis revealed that our single-factor SOX2 TD strategy converted human skin fibroblasts into h-iNSCTE that were nestin+ and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSCTE rapidly migrated to human GBM cells and penetrated human GBM spheroids, a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSCTE delivery of the proapoptotic agent tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) reduced the size of solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. Additionally, h-iNSCTE thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSCTE-TK) reduced the size of patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical NSC therapy, h-iNSCTE-TK therapy delivered into the postoperative surgical resection cavity delayed the regrowth of residual GBMs threefold and prolonged survival from 46 to 60 days. These results suggest that TD of human skin into h-iNSCTE is a platform for creating tumor-homing cytotoxic cell therapies for cancer, where the potential to avoid carrier rejection could maximize treatment durability in human trials.
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Affiliation(s)
- Juli R Bagó
- Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Onyi Okolie
- Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Raluca Dumitru
- UNC Human Pluripotent Stem Cell Core Facility, Department of Genetics, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Matthew G Ewend
- Department of Neurosurgery, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Joel S Parker
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Ryan Vander Werff
- Department of Cellular and Physiological Sciences, Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
| | - T Michael Underhill
- Department of Cellular and Physiological Sciences, Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
| | - Ralf S Schmid
- Division of Neuropathology and Department of Pathology and Laboratory Medicine, Department of Neurology and Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - C Ryan Miller
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Division of Neuropathology and Department of Pathology and Laboratory Medicine, Department of Neurology and Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Shawn D Hingtgen
- Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. .,Department of Neurosurgery, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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Kwon D, Ahn HJ, Kang KS. Generation of Human Neural Stem Cells by Direct Phenotypic Conversion. Results Probl Cell Differ 2018; 66:103-121. [PMID: 30209656 DOI: 10.1007/978-3-319-93485-3_4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
Human neural stem cells (hNSC) are multipotent adult stem cells. Various studies are underway worldwide to identify new methods for treatment of neurological diseases using hNSC. This chapter summarizes the latest research trends in and fields for application of patient-specific hNSC using direct phenotypic conversion technology. The aim of the study was to analyze the advantages and disadvantages of current technology and to suggest relevant directions for future hNSC research.
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Affiliation(s)
- Daekee Kwon
- Stem Cells and Regenerative Bioengineering Institute in Kangstem Biotech, Seoul National University, Seoul, South Korea
| | - Hee-Jin Ahn
- Stem Cells and Regenerative Bioengineering Institute in Kangstem Biotech, Seoul National University, Seoul, South Korea
| | - Kyung-Sun Kang
- Stem Cells and Regenerative Bioengineering Institute in Kangstem Biotech, Seoul National University, Seoul, South Korea.
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, South Korea.
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28
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Connor B. Concise Review: The Use of Stem Cells for Understanding and Treating Huntington's Disease. Stem Cells 2017; 36:146-160. [PMID: 29178352 DOI: 10.1002/stem.2747] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2017] [Accepted: 10/13/2017] [Indexed: 12/20/2022]
Abstract
Two decades ago, researchers identified that a CAG expansion mutation in the huntingtin (HTT) gene was involved in the pathogenesis of Huntington's disease (HD). However, since the identification of the HTT gene, there has been no advance in the development of therapeutic strategies to prevent or reduce the progression of HD. With the recent advances in stem cell biology and human cell reprogramming technologies, several novel and exciting pathways have emerged allowing researchers to enhance their understanding of the pathogenesis of HD, to identify and screen potential drug targets, and to explore alternative donor cell sources for cell replacement therapy. This review will discuss the role of compensatory neurogenesis in the HD brain, the use of stem cell-based therapies for HD to replace or prevent cell loss, and the recent advance of cell reprogramming to model and/or treat HD. These new technologies, coupled with advances in genome editing herald a promising new era for HD research with the potential to identify a therapeutic strategy to alleviate this debilitating disorder. Stem Cells 2018;36:146-160.
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Affiliation(s)
- Bronwen Connor
- Department of Pharmacology and Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
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29
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Playne R, Connor B. Understanding Parkinson's Disease through the Use of Cell Reprogramming. Stem Cell Rev Rep 2017; 13:151-169. [PMID: 28083784 DOI: 10.1007/s12015-017-9717-5] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Recent progress in the field of somatic cell reprogramming offers exciting new possibilities for the study and treatment of Parkinson's disease (PD). Reprogramming technology offers the ability to untangle the diverse contributing risk factors for PD, such as ageing, genetics and environmental toxins. In order to gain novel insights into such a complex disease, cell-based models of PD should represent, as closely as possible, aged human dopaminergic neurons of the substantia nigra. However, the generation of high yields of functionally mature, authentic ventral midbrain dopamine (vmDA) neurons has not been easy to achieve. Furthermore, ensuring cells represent aged rather than embryonic neurons has presented a significant challenge. To date, induced pluripotent stem (iPS) cells have received much attention for modelling PD. Nonetheless, direct reprogramming strategies (either to a neuronal or neural stem/progenitor fate) represent a valid alternative that are yet to be extensively explored. Direct reprogramming is faster and more efficient than iPS cell reprogramming, and appears to conserve age-related markers. At present, however, protocols aiming to derive authentic, mature vmDA neurons by direct reprogramming of adult human somatic cells are sorely lacking. This review will discuss the strategies that have been employed to generate vmDA neurons and their potential for the study and treatment of PD.
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Affiliation(s)
- Rebecca Playne
- Department of Pharmacology and Clinical Pharmacology, Centre for Brain Research, School of Medical Science, FMHS, University of Auckland, Auckland, 1023, New Zealand
| | - Bronwen Connor
- Department of Pharmacology and Clinical Pharmacology, Centre for Brain Research, School of Medical Science, FMHS, University of Auckland, Auckland, 1023, New Zealand.
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30
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López-León M, Outeiro TF, Goya RG. Cell reprogramming: Therapeutic potential and the promise of rejuvenation for the aging brain. Ageing Res Rev 2017; 40:168-181. [PMID: 28903069 DOI: 10.1016/j.arr.2017.09.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2017] [Revised: 08/27/2017] [Accepted: 09/05/2017] [Indexed: 02/06/2023]
Abstract
Aging is associated with a progressive increase in the incidence of neurodegenerative diseases, with Alzheimer's (AD) and Parkinson's (PD) disease being the most conspicuous examples. Within this context, the absence of efficacious therapies for most age-related brain pathologies has increased the interest in regenerative medicine. In particular, cell reprogramming technologies have ushered in the era of personalized therapies that not only show a significant potential for the treatment of neurodegenerative diseases but also promise to make biological rejuvenation feasible. We will first review recent evidence supporting the emerging view that aging is a reversible epigenetic phenomenon. Next, we will describe novel reprogramming approaches that overcome some of the intrinsic limitations of conventional induced-pluripotent-stem-cell technology. One of the alternative approaches, lineage reprogramming, consists of the direct conversion of one adult cell type into another by transgenic expression of multiple lineage-specific transcription factors (TF). Another strategy, termed pluripotency factor-mediated direct reprogramming, uses universal TF to generate epigenetically unstable intermediates able to differentiate into somatic cell types in response to specific differentiation factors. In the third part we will review studies showing the potential relevance of the above approaches for the treatment of AD and PD.
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Affiliation(s)
- Micaela López-León
- Institute for Biochemical Research (INIBIOLP) - Histology B & Pathology B, School of Medicine, National University of La Plata, La Plata, Argentina
| | - Tiago F Outeiro
- Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany; Max Planck Institute for Experimental Medicine, Göttingen, Germany
| | - Rodolfo G Goya
- Institute for Biochemical Research (INIBIOLP) - Histology B & Pathology B, School of Medicine, National University of La Plata, La Plata, Argentina.
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31
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Khazaei N, Rastegar-Pouyani S, O'Toole N, Wee P, Mohammadnia A, Yaqubi M. Regulating the transcriptomes that mediate the conversion of fibroblasts to various nervous system neural cell types. J Cell Physiol 2017; 233:3603-3614. [PMID: 29044560 DOI: 10.1002/jcp.26221] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Accepted: 10/05/2017] [Indexed: 12/31/2022]
Abstract
Our understanding of the mechanism of cell fate transition during the direct reprogramming of fibroblasts into various central nervous system (CNS) neural cell types has been limited by the lack of a comprehensive analysis on generated cells, independently and in comparison with other CNS neural cell types. Here, we applied an integrative approach on 18 independent high throughput expression data sets to gain insight into the regulation of the transcriptome during the conversion of fibroblasts into induced neural stem cells, induced neurons (iNs), induced astrocytes, and induced oligodendrocyte progenitor cells (iOPCs). We found common down-regulated genes to be mostly related to fibroblast-specific functions, and suggest their potential as markers for screening of the silencing of the fibroblast-specific program. For example, Tagln was significantly down-regulated across all considered data sets. In addition, we identified specific profiles of up-regulated genes for each CNS neural cell types, which could be potential markers for maturation and efficiency screenings. Furthermore, we identified the main TFs involved in the regulation of the gene expression program during direct reprogramming. For example, in the generation of iNs from fibroblasts, the Rest TF was the main regulator of this reprogramming. In summary, our computational approach for meta-analyzing independent expression data sets provides significant details regarding the molecular mechanisms underlying the regulation of the gene expression program, and also suggests potentially useful candidate genes for screening down-regulation of fibroblast gene expression profile, maturation, and efficiency, as well as candidate TFs for increasing the efficiency of the reprogramming process.
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Affiliation(s)
- Niusha Khazaei
- Meakins-Christie Laboratories, Department of Medicine, McGill University and McGill University Health Centre Research Institute, Montréal, Canada
| | | | - Nicholas O'Toole
- Douglas Mental Health University Institute, McGill University, Ludmer Centre for Neuroinformatics and Mental Health Montreal, Quebec, Canada
| | - Ping Wee
- Faculty of Medicine and Dentistry, Department of Medical Genetics and Signal Transduction Research Group, University of Alberta, Edmonton, Alberta, Canada
| | | | - Moein Yaqubi
- Department of Psychiatry, Sackler Program for Epigenetics and Psychobiology at McGill University, Ludmer Centre for Neuroinformatics and Mental Health, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada
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Rastegar-Pouyani S, Khazaei N, Wee P, Mohammadnia A, Yaqubi M. Role of Hepatic-Specific Transcription Factors and Polycomb Repressive Complex 2 during Induction of Fibroblasts to Hepatic Fate. PLoS One 2016; 11:e0167081. [PMID: 27902735 PMCID: PMC5130264 DOI: 10.1371/journal.pone.0167081] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2016] [Accepted: 11/08/2016] [Indexed: 01/08/2023] Open
Abstract
Direct reprogramming using defined sets of transcription factors (TFs) is a recent strategy for generating induced hepatocytes (iHeps) from fibroblasts for use in regenerative medicine and drug development. Comprehensive studies detailing the regulatory role of TFs during this reprogramming process could help increase its efficiency. This study aimed to find the TFs with the greatest influences on the generation of iHeps from fibroblasts, and to further understand their roles in the regulation of the gene expression program. Here, we used systems biology approaches to analyze high quality expression data sets in combination with TF-binding sites data and protein-protein interactions data during the direct reprogramming of fibroblasts to iHeps. Our results revealed two main patterns for differentially expressed genes (DEGs): up-regulated genes were categorized as hepatic-specific pattern, and down-regulated genes were categorized as mesoderm- and fibroblast-specific pattern. Interestingly, hepatic-specific genes co-expressed and were regulated by hepatic-specific TFs, specifically Hnf4a and Foxa2. Conversely, the mesoderm- and fibroblast-specific pattern was mainly silenced by polycomb repressive complex 2 (PRC2) members, including Suz12, Mtf2, Ezh2, and Jarid2. Independent analysis of both the gene and core regulatory network of DE-TFs showed significant roles for Hnf4a, Foxa2, and PRC2 members in the regulation of the gene expression program and in biological processes during the direct conversion process. Altogether, using systems biology approaches, we clarified the role of Hnf4a and Foxa2 as hepatic-specific TFs, and for the first time, introduced the PRC2 complex as the main regulator that favors the direct reprogramming process in cooperation with hepatic-specific factors.
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Affiliation(s)
- Shima Rastegar-Pouyani
- Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
| | - Niusha Khazaei
- Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
| | - Ping Wee
- Department of Medical Genetics and Signal Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
| | - Abdulshakour Mohammadnia
- Department of Human Genetics, Division of Hematology and Oncology, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
| | - Moein Yaqubi
- Ludmer Centre for Neuroinformatics and Mental Health, McGill University, Montreal, Quebec, Canada
- Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada
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Kwon D, Ji M, Lee S, Seo KW, Kang KS. Reprogramming Enhancers in Somatic Cell Nuclear Transfer, iPSC Technology, and Direct Conversion. Stem Cell Rev Rep 2016; 13:24-34. [DOI: 10.1007/s12015-016-9697-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
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Analysis of induced pluripotent stem cells carrying 22q11.2 deletion. Transl Psychiatry 2016; 6:e934. [PMID: 27801899 PMCID: PMC5314118 DOI: 10.1038/tp.2016.206] [Citation(s) in RCA: 75] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/06/2015] [Revised: 08/24/2016] [Accepted: 08/25/2016] [Indexed: 12/18/2022] Open
Abstract
Given the complexity and heterogeneity of the genomic architecture underlying schizophrenia, molecular analyses of these patients with defined and large effect-size genomic defects could provide valuable clues. We established human-induced pluripotent stem cells from two schizophrenia patients with the 22q11.2 deletion (two cell lines from each subject, total of four cell lines) and three controls (total of four cell lines). Neurosphere size, neural differentiation efficiency, neurite outgrowth, cellular migration and the neurogenic-to-gliogenic competence ratio were significantly reduced in patient-derived cells. As an underlying mechanism, we focused on the role of DGCR8, a key gene for microRNA (miRNA) processing and mapped in the deleted region. In mice, Dgcr8 hetero-knockout is known to show a similar phenotype of reduced neurosphere size (Ouchi et al., 2013). The miRNA profiling detected reduced expression levels of miRNAs belonging to miR-17/92 cluster and miR-106a/b in the patient-derived neurospheres. Those miRNAs are reported to target p38α, and conformingly the levels of p38α were upregulated in the patient-derived cells. p38α is known to drive gliogenic differentiation. The inhibition of p38 activity by SB203580 in patient-derived neurospheres partially restored neurogenic competence. Furthermore, we detected elevated expression of GFAP, a gliogenic (astrocyte) marker, in postmortem brains from schizophrenia patients without the 22q11.2 deletion, whereas inflammation markers (IL1B and IL6) remained unchanged. In contrast, a neuronal marker, MAP2 expressions were decreased in schizophrenia brains. These results suggest that a dysregulated balance of neurogenic-to-gliogenic competence may underlie neurodevelopmental disorders such as schizophrenia.
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35
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Engineering cell fate: Spotlight on cell-activation and signaling-directed lineage conversion. Tissue Cell 2016; 48:475-87. [DOI: 10.1016/j.tice.2016.07.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2016] [Revised: 06/13/2016] [Accepted: 07/25/2016] [Indexed: 12/23/2022]
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Kim J, Henley BM, Kim CH, Lester HA, Yang C. Incubator embedded cell culture imaging system (EmSight) based on Fourier ptychographic microscopy. BIOMEDICAL OPTICS EXPRESS 2016; 7:3097-110. [PMID: 27570701 PMCID: PMC4986817 DOI: 10.1364/boe.7.003097] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/23/2016] [Revised: 07/07/2016] [Accepted: 07/17/2016] [Indexed: 05/20/2023]
Abstract
Multi-day tracking of cells in culture systems can provide valuable information in bioscience experiments. We report the development of a cell culture imaging system, named EmSight, which incorporates multiple compact Fourier ptychographic microscopes with a standard multiwell imaging plate. The system is housed in an incubator and presently incorporates six microscopes. By using the same low magnification objective lenses as the objective and the tube lens, the EmSight is configured as a 1:1 imaging system that, providing large field-of-view (FOV) imaging onto a low-cost CMOS imaging sensor. The EmSight improves the image resolution by capturing a series of images of the sample at varying illumination angles; the instrument reconstructs a higher-resolution image by using the iterative Fourier ptychographic algorithm. In addition to providing high-resolution brightfield and phase imaging, the EmSight is also capable of fluorescence imaging at the native resolution of the objectives. We characterized the system using a phase Siemens star target, and show four-fold improved coherent resolution (synthetic NA of 0.42) and a depth of field of 0.2 mm. To conduct live, long-term dopaminergic neuron imaging, we cultured ventral midbrain from mice driving eGFP from the tyrosine hydroxylase promoter. The EmSight system tracks movements of dopaminergic neurons over a 21 day period.
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Affiliation(s)
- Jinho Kim
- Department of Electrical Engineering, California Institute of Technology, Pasadena, California 91125, USA
| | - Beverley M. Henley
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA
| | - Charlene H. Kim
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA
| | - Henry A. Lester
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA
| | - Changhuei Yang
- Department of Electrical Engineering, California Institute of Technology, Pasadena, California 91125, USA
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Harada K, Ferdous T, Cui D, Kuramitsu Y, Matsumoto T, Ikeda E, Okano H, Ueyama Y. Induction of artificial cancer stem cells from tongue cancer cells by defined reprogramming factors. BMC Cancer 2016; 16:548. [PMID: 27464948 PMCID: PMC4963932 DOI: 10.1186/s12885-016-2416-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Accepted: 06/15/2016] [Indexed: 12/30/2022] Open
Abstract
Background The cancer stem cells (CSCs), a small subpopulation of cells in tumor are responsible for the tumor initiation, growth, recurrence and metastasis of cancer, as well as resistance of cancers to drugs or radiotherapy. CSCs are an important target for the development of novel strategies in cancer treatment. However, CSCs-targeted new anti-cancer drug discovery is currently hindered by the lack of easy and reliable methods for isolating, collecting and maintaining sufficient number of CSCs. Here, we examined whether introduction of defined reprogramming factors (Oct4, shp53, Sox2, Klf4, l-Myc and Lin28) into HSC2 tongue cancer cells could transform the HSC2 into HSC2 with CSCs properties. Methods We introduced the defined reprogramming factors into HSC2 tongue cancer cells via episomal vectors by electroporation method to generate transfectant cells. We investigated the malignant properties of the transfectant cells by cell proliferation assay, migration assay, wound healing assay, sphere formation assay, chemosensitivity and radiosensitivity assay in vitro; and also examined the tumorigenic potential of the transfectants in vivo. Results The transfectant cells (HSC2/hOCT3/4-shp53-F, HSC2/hSK, HSC2/hUL, HSC2/hOCT3/4-shp53-F + hSK, HSC2/hOCT3/4-shp53-F + hUL, HSC2/hSK + hUL, HSC2/hOCT3/4-shp53-F + hSK + hUL) displayed a malignant phenotype in culture and form tumors on the back of nude mice more efficiently than parental HSC2 and control HSC2/EGFP transfectant cells. They exhibited increased resistance to chemotherapeutic agents; 5-fluorouracil, cisplatin, docetaxel, trifluorothymidine, zoledronic acid, cetuximab, bortezomib and radiation when compared with HSC2 and HSC2/EGFP. Among all the transfected cells, HSC2/hOCT3/4-shp53-F + hSK + hUL cell containing all of the reprogramming factors showed the most aggressive and malignant properties and presented the highest number of spheres in the culture medium containing human recombinant fibroblast Growth Factor-2 (FGF-2) and epidermal Growth Factor (EGF). Conclusion These findings suggest that artificial cancer stem cells obtained by the induction of cellular reprogramming may be useful for investigating the acquisition of potential malignancy as well as screening the CSCs-targeting drugs.
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Affiliation(s)
- Koji Harada
- Department of Oral and Maxillofacial Surgery, Yamaguchi University Graduate School of Medicine, 1-1-1, Minamikogushi, Ube, 755-8505, Japan.
| | - Tarannum Ferdous
- Department of Oral and Maxillofacial Surgery, Yamaguchi University Graduate School of Medicine, 1-1-1, Minamikogushi, Ube, 755-8505, Japan
| | - Dan Cui
- Department of Pathology, Yamaguchi University Graduate School of Medicine, 1-1-1, Minamikogushi, Ube, 755-8505, Japan
| | - Yasuhiro Kuramitsu
- Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, 1-1-1, Minamikogushi, Ube, 755-8505, Japan
| | - Takuya Matsumoto
- Department of Physiology, Keio University, School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Eiji Ikeda
- Department of Pathology, Yamaguchi University Graduate School of Medicine, 1-1-1, Minamikogushi, Ube, 755-8505, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University, School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Yoshiya Ueyama
- Department of Oral and Maxillofacial Surgery, Yamaguchi University Graduate School of Medicine, 1-1-1, Minamikogushi, Ube, 755-8505, Japan
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Petersen GF, Strappe PM. Generation of diverse neural cell types through direct conversion. World J Stem Cells 2016; 8:32-46. [PMID: 26981169 PMCID: PMC4766249 DOI: 10.4252/wjsc.v8.i2.32] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Revised: 11/18/2015] [Accepted: 01/04/2016] [Indexed: 02/06/2023] Open
Abstract
A characteristic of neurological disorders is the loss of critical populations of cells that the body is unable to replace, thus there has been much interest in identifying methods of generating clinically relevant numbers of cells to replace those that have been damaged or lost. The process of neural direct conversion, in which cells of one lineage are converted into cells of a neural lineage without first inducing pluripotency, shows great potential, with evidence of the generation of a range of functional neural cell types both in vitro and in vivo, through viral and non-viral delivery of exogenous factors, as well as chemical induction methods. Induced neural cells have been proposed as an attractive alternative to neural cells derived from embryonic or induced pluripotent stem cells, with prospective roles in the investigation of neurological disorders, including neurodegenerative disease modelling, drug screening, and cellular replacement for regenerative medicine applications, however further investigations into improving the efficacy and safety of these methods need to be performed before neural direct conversion becomes a clinically viable option. In this review, we describe the generation of diverse neural cell types via direct conversion of somatic cells, with comparison against stem cell-based approaches, as well as discussion of their potential research and clinical applications.
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Nakajima-Koyama M, Lee J, Ohta S, Yamamoto T, Nishida E. Induction of Pluripotency in Astrocytes through a Neural Stem Cell-like State. J Biol Chem 2015; 290:31173-88. [PMID: 26553868 DOI: 10.1074/jbc.m115.683466] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2015] [Indexed: 01/20/2023] Open
Abstract
It remains controversial whether the routes from somatic cells to induced pluripotent stem cells (iPSCs) are related to the reverse order of normal developmental processes. Specifically, it remains unaddressed whether or not the differentiated cells become iPSCs through their original tissue stem cell-like state. Previous studies analyzing the reprogramming process mostly used fibroblasts; however, the stem cell characteristics of fibroblasts made it difficult to address this. Here, we generated iPSCs from mouse astrocytes, a type of glial cells, by three (OCT3/4, KLF4, and SOX2), two (OCT3/4 and KLF4), or four (OCT3/4, KLF4, and SOX2 plus c-MYC) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently up-regulated during reprogramming, and Sox1-positive cells become iPSCs. The up-regulation of Sox1 is essential for OCT3/4- and KLF4-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during fibroblast reprogramming, neither Sox1 up-regulation nor an increase in neurogenic potential occurs. Our results thus demonstrate that astrocytes are reprogrammed through an NSC-like state.
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Affiliation(s)
- May Nakajima-Koyama
- From the Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, CREST, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan
| | - Joonseong Lee
- From the Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502
| | - Sho Ohta
- From the Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, the Department of Reprogramming Science, Center for iPS Cell Research and Application, and
| | - Takuya Yamamoto
- CREST, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan the Department of Reprogramming Science, Center for iPS Cell Research and Application, and Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Sakyo-ku, Kyoto 606-8507, and
| | - Eisuke Nishida
- From the Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, CREST, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan
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Karumbaiah L, Enam SF, Brown AC, Saxena T, Betancur MI, Barker TH, Bellamkonda RV. Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Niches for Neural Stem Cells. Bioconjug Chem 2015; 26:2336-49. [PMID: 26440046 DOI: 10.1021/acs.bioconjchem.5b00397] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Neural stem cells (NSCs) possess great potential for neural tissue repair after traumatic injuries to the central nervous system (CNS). However, poor survival and self-renewal of NSCs after injury severely limits its therapeutic potential. Sulfated chondroitin sulfate glycosaminoglycans (CS-GAGs) linked to CS proteoglycans (CSPGs) in the brain extracellular matrix (ECM) have the ability to bind and potentiate trophic factor efficacy, and promote NSC self-renewal in vivo. In this study, we investigated the potential of CS-GAG hydrogels composed of monosulfated CS-4 (CS-A), CS-6 (CS-C), and disulfated CS-4,6 (CS-E) CS-GAGs as NSC carriers, and their ability to create endogenous niches by enriching specific trophic factors to support NSC self-renewal. We demonstrate that CS-GAG hydrogel scaffolds showed minimal swelling and degradation over a period of 15 days in vitro, absorbing only 6.5 ± 0.019% of their initial weight, and showing no significant loss of mass during this period. Trophic factors FGF-2, BDNF, and IL10 bound with high affinity to CS-GAGs, and were significantly (p < 0.05) enriched in CS-GAG hydrogels when compared to unsulfated hyaluronic acid (HA) hydrogels. Dissociated rat subventricular zone (SVZ) NSCs when encapsulated in CS-GAG hydrogels demonstrated ∼88.5 ± 6.1% cell viability in vitro. Finally, rat neurospheres in CS-GAG hydrogels conditioned with the mitogen FGF-2 demonstrated significantly (p < 0.05) higher self-renewal when compared to neurospheres cultured in unconditioned hydrogels. Taken together, these findings demonstrate the ability of CS-GAG based hydrogels to regulate NSC self-renewal, and facilitate growth factor enrichment locally.
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Affiliation(s)
- Lohitash Karumbaiah
- Regenerative Bioscience Center, ADS Complex, The University of Georgia , 425 River Road, Athens, Georgia 30602, United States
| | | | - Ashley C Brown
- Joint Department of Biomedical Engineering NC State University/UNC-Chapel Hill , 4204 B Engineering Building III, Raleigh, North Carolina 27695, United States
| | | | - Martha I Betancur
- Regenerative Bioscience Center, ADS Complex, The University of Georgia , 425 River Road, Athens, Georgia 30602, United States
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Histone modifications controlling native and induced neural stem cell identity. Curr Opin Genet Dev 2015; 34:95-101. [DOI: 10.1016/j.gde.2015.08.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2015] [Revised: 07/31/2015] [Accepted: 08/11/2015] [Indexed: 12/31/2022]
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Transdifferentiation-Induced Neural Stem Cells Promote Recovery of Middle Cerebral Artery Stroke Rats. PLoS One 2015; 10:e0137211. [PMID: 26352672 PMCID: PMC4564190 DOI: 10.1371/journal.pone.0137211] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Accepted: 08/14/2015] [Indexed: 01/19/2023] Open
Abstract
Induced neural stem cells (iNSCs) can be directly transdifferentiated from somatic cells. One potential clinical application of the iNSCs is for nerve regeneration. However, it is unknown whether iNSCs function in disease models. We produced transdifferentiated iNSCs by conditional overexpressing Oct4, Sox2, Klf4, c-Mycin mouse embryonic fibroblasts. They expanded readily in vitro and expressed NSC mRNA profile and protein markers. These iNSCs differentiated into mature astrocytes, neurons and oligodendrocytes in vitro. Importantly, they reduced lesion size, promoted the recovery of motor and sensory function as well as metabolism status in middle cerebral artery stroke rats. These iNSCs secreted nerve growth factors, which was associated with observed protection of neurons from apoptosis. Furthermore, iNSCs migrated to and passed through the lesion in the cerebral cortex, where Tuj1+ neurons were detected. These findings have revealed the function of transdifferentiated iNSCs in vivo, and thus provide experimental evidence to support the development of personalized regenerative therapy for CNS diseases by using genetically engineered autologous somatic cells.
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Mozafari S, Laterza C, Roussel D, Bachelin C, Marteyn A, Deboux C, Martino G, Baron-Van Evercooren A. Skin-derived neural precursors competitively generate functional myelin in adult demyelinated mice. J Clin Invest 2015; 125:3642-56. [PMID: 26301815 DOI: 10.1172/jci80437] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2015] [Accepted: 07/07/2015] [Indexed: 12/26/2022] Open
Abstract
Induced pluripotent stem cell-derived (iPS-derived) neural precursor cells may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin diseases. So far, the therapeutic potential of reprogrammed cells has been evaluated in neonatal demyelinating models. However, the repair efficacy and safety of these cells has not been well addressed in the demyelinated adult CNS, which has decreased cell plasticity and scarring. Moreover, it is not clear if these induced pluripotent-derived cells have the same reparative capacity as physiologically committed CNS-derived precursors. Here, we performed a side-by-side comparison of CNS-derived and skin-derived neural precursors in culture and following engraftment in murine models of adult spinal cord demyelination. Grafted induced neural precursors exhibited a high capacity for survival, safe integration, migration, and timely differentiation into mature bona fide oligodendrocytes. Moreover, grafted skin-derived neural precursors generated compact myelin around host axons and restored nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Together, these results provide important insights into the biology of reprogrammed cells in adult demyelinating conditions and support use of these cells for regenerative biomedicine of myelin diseases that affect the adult CNS.
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Mirakhori F, Zeynali B, Rassouli H, Shahbazi E, Hashemizadeh S, Kiani S, Salekdeh GH, Baharvand H. Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach. PLoS One 2015; 10:e0135479. [PMID: 26266943 PMCID: PMC4534403 DOI: 10.1371/journal.pone.0135479] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2014] [Accepted: 07/22/2015] [Indexed: 12/19/2022] Open
Abstract
BACKGROUND A number of studies generated induced neural progenitor cells (iNPCs) from human fibroblasts by viral delivering defined transcription factors. However, the potential risks associated with gene delivery systems have limited their clinical use. We propose it would be safer to induce neural progenitor-like cells from human adult fibroblasts via a direct non-genetic alternative approach. METHODOLOGY/PRINCIPAL FINDINGS Here, we have reported that seven rounds of TAT-SOX2 protein transduction in a defined chemical cocktail under a 3D sphere culture gradually morphed fibroblasts into neuroepithelial-like colonies. We were able to expand these cells for up to 20 passages. These cells could give rise to cells that expressed neurons and glia cell markers both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE These results show that our approach is beneficial for the genetic material-free generation of iNPCs from human fibroblasts where small chemical molecules can provide a valuable, viable strategy to boost and improve induction in a 3D sphere culture.
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Affiliation(s)
- Fahimeh Mirakhori
- School of Biology, College of Science, University of Tehran, Tehran, Iran
- Department of Stem cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Bahman Zeynali
- School of Biology, College of Science, University of Tehran, Tehran, Iran
| | - Hassan Rassouli
- Department of Stem cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Ebrahim Shahbazi
- Department of Stem cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Shiva Hashemizadeh
- Department of Stem cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Sahar Kiani
- Department of Stem cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Ghasem Hosseini Salekdeh
- Department of Molecular Systems Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hossein Baharvand
- Department of Stem cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
- Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran
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Utility of Scalp Hair Follicles as a Novel Source of Biomarker Genes for Psychiatric Illnesses. Biol Psychiatry 2015; 78:116-25. [PMID: 25444170 DOI: 10.1016/j.biopsych.2014.07.025] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/17/2014] [Revised: 07/24/2014] [Accepted: 07/25/2014] [Indexed: 12/26/2022]
Abstract
BACKGROUND Identifying beneficial surrogate genetic markers in psychiatric disorders is crucial but challenging. METHODS Given that scalp hair follicles are easily accessible and, like the brain, are derived from the ectoderm, expressions of messenger RNA (mRNA) and microRNA in the organ were examined between schizophrenia (n for first/second = 52/42) and control subjects (n = 62/55) in two sets of cohort. Genes of significance were also analyzed using postmortem brains (n for case/control = 35/35 in Brodmann area 46, 20/20 in cornu ammonis 1) and induced pluripotent stem cells (n = 4/4) and pluripotent stem cell-derived neurospheres (n = 12/12) to see their role in the central nervous system. Expression levels of mRNA for autism (n for case/control = 18/24) were also examined using scalp hair follicles. RESULTS Among mRNA examined, FABP4 was downregulated in schizophrenia subjects by two independent sample sets. Receiver operating characteristic curve analysis determined that the sensitivity and specificity were 71.8% and 66.7%, respectively. FABP4 was expressed from the stage of neurosphere. Additionally, microarray-based microRNA analysis showed a trend of increased expression of hsa-miR-4449 (p = .0634) in hair follicles from schizophrenia. hsa-miR-4449 expression was increased in Brodmann area 46 from schizophrenia (p = .0007). Finally, we tested the expression of nine putative autism candidate genes in hair follicles and found decreased CNTNAP2 expression in the autism cohort. CONCLUSIONS Scalp hair follicles could be a beneficial genetic biomarker resource for brain diseases, and further studies of FABP4 are merited in schizophrenia pathogenesis.
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Lim MS, Chang MY, Kim SM, Yi SH, Suh-Kim H, Jung SJ, Kim MJ, Kim JH, Lee YS, Lee SY, Kim DW, Lee SH, Park CH. Generation of Dopamine Neurons from Rodent Fibroblasts through the Expandable Neural Precursor Cell Stage. J Biol Chem 2015; 290:17401-14. [PMID: 26023233 DOI: 10.1074/jbc.m114.629808] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2014] [Indexed: 01/08/2023] Open
Abstract
Recent groundbreaking work has demonstrated that combined expression of the transcription factors Brn2, Ascl1, and Myt1L (BAM; also known as Wernig factors) convert mouse fibroblasts into postmitotic neuronal cells. However, questions remain regarding whether trans-conversion is achieved directly or involves an intermediary precursor stage. Trans-conversion toward expandable neural precursor cells (NPCs) is more useful than direct one-step neuron formation with respect to yielding a sufficient number of cells and the feasibility of manipulating NPC differentiation toward certain neuron subtypes. Here, we show that co-expression of Wernig factors and Bcl-xL induces fibroblast conversion into NPCs (induced NPCs (iNPCs)) that are highly expandable for >100 passages. Gene expression analyses showed that the iNPCs exhibited high expression of common NPC genes but not genes specific to defined embryonic brain regions. This finding indicated that a regional identity of iNPCs was not established. Upon induction, iNPCs predominantly differentiated into astrocytes. However, the differentiation potential was not fixed and could be efficiently manipulated into general or specific subtypes of neurons by expression of additional genes. Specifically, overexpression of Nurr1 and Foxa2, transcription factors specific for midbrain dopamine neuron development, drove iNPCs to yield mature midbrain dopamine neurons equipped with presynaptic DA neuronal functions. We further assessed the therapeutic potential of iNPCs in Parkinson disease model rats.
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Affiliation(s)
- Mi-Sun Lim
- From the Graduate School of Biomedical Science and Engineering, the Hanyang Biomedical Research Institute, the Departments of Microbiology
| | - Mi-Yoon Chang
- the Hanyang Biomedical Research Institute, Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul 133-791, Korea
| | - Sang-Mi Kim
- the Department of Biomedical Science, Graduate School, and
| | - Sang-Hoon Yi
- the Hanyang Biomedical Research Institute, Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul 133-791, Korea
| | - Haeyoung Suh-Kim
- the Department of Anatomy and Brain Disease Research Center, College of Medicine, Ajou University, Suwon 443-749, Korea, and
| | - Sung Jun Jung
- the Hanyang Biomedical Research Institute, Physiology, and
| | - Min Jung Kim
- the Hanyang Biomedical Research Institute, Physiology, and
| | - Jin Hyuk Kim
- From the Graduate School of Biomedical Science and Engineering, the Hanyang Biomedical Research Institute, Physiology, and
| | - Yong-Sung Lee
- From the Graduate School of Biomedical Science and Engineering, the Hanyang Biomedical Research Institute, Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul 133-791, Korea
| | | | - Dong-Wook Kim
- the Department of Physiology and Cell Therapy Center, Yonsei University College of Medicine, Seoul 120-752, Korea
| | - Sang-Hun Lee
- the Hanyang Biomedical Research Institute, Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul 133-791, Korea,
| | - Chang-Hwan Park
- From the Graduate School of Biomedical Science and Engineering, the Hanyang Biomedical Research Institute, the Departments of Microbiology,
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Sakamoto R, Rahman MM, Shimomura M, Itoh M, Nakatsura T. Time-lapse imaging assay using the BioStation CT: a sensitive drug-screening method for three-dimensional cell culture. Cancer Sci 2015; 106:757-765. [PMID: 25865675 PMCID: PMC4471796 DOI: 10.1111/cas.12667] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2015] [Revised: 03/27/2015] [Accepted: 03/31/2015] [Indexed: 12/24/2022] Open
Abstract
Three-dimensional (3D) cell culture is beneficial for physiological studies of tumor cells, due to its potential to deliver a high quantity of cell culture information that is representative of the cancer microenvironment and predictive of drug responses in vivo. Currently, gel-associated or matrix-associated 3D cell culture is comprised of intricate procedures that often result in experimental complexity. Therefore, we developed an innovative anti-cancer drug sensitivity screening technique for 3D cell culture on NanoCulture Plates (NCP) by employing the imaging device BioStation CT. Here, we showed that the human breast cancer cell lines BT474 and T47D form multicellular spheroids on NCP plates and compared their sensitivity to the anti-cancer drugs trastuzumab and paclitaxel using the BioStation CT. The anticancer drugs reduced spheroid migration velocity and suppressed spheroid fusion. In addition, primary cells derived from the human breast cancer tissues B58 and B61 grown on NCP plates also exhibited similar drug sensitivity. These results were in good agreement with the conventional assay method using ATP quantification. We confirmed the antitumor effects of the drugs on cells seeded in 96-well plates using the BioStation CT imaging technique. We expect this method to be useful in research for new antitumor agents and for drug sensitivity tests in individually-tailored cancer treatments.
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Affiliation(s)
| | | | - Manami Shimomura
- Division of Cancer Immunotherapy, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, Chiba, Japan
| | - Manabu Itoh
- SCIVAX Life Sciences, Kawasaki, Kanagawa, Japan
| | - Tetsuya Nakatsura
- Division of Cancer Immunotherapy, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, Chiba, Japan
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Yaqubi M, Mohammadnia A, Fallahi H. Predicting involvement of polycomb repressive complex 2 in direct conversion of mouse fibroblasts into induced neural stem cells. Stem Cell Res Ther 2015; 6:42. [PMID: 25890371 PMCID: PMC4397673 DOI: 10.1186/s13287-015-0045-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2014] [Revised: 01/24/2015] [Accepted: 03/09/2015] [Indexed: 11/19/2022] Open
Abstract
Introduction Mouse fibroblasts could be directly converted into induced neural stem cells (iNSCs), by introducing a set of known transcription factors (TFs). This process, known as direct reprogramming, is an alternative source of NSCs production for cell therapy applications, hence, more common sources for such cells including embryonic stem cell (ESCs) and induced pluripotent stem cell (iPSCs) are also in use. Despite their importance, the exact role of different TFs involved in the conversion of fibroblasts into iNSCs and the interactions between these factors has not been studied. Methods Here, we have used available microarray data to construct a gene regulatory network to understand the dynamic of regulatory interactions during this conversion. We have implemented other types of data such as information regarding TFs binding sites and valid protein-protein interactions to improve the network reliability. The network contained 1857 differentially expressed (DE) genes, linked by11054 interactions. The most important TFs identified based on topology analysis of the network. Furthermore, in selecting such TFs, we have also considered their role in the regulation of nervous system development. Results Based on these analyses, we found that Ezh2, Jarid2, Mtf2, Nanog, Pou5f1, Sall4, Smarca4, Sox2, Suz12, and Tcf3 are the main regulators of direct conversion of mouse fibroblasts into iNSCs. Because, members of the polycomb repressive complex 2 (PRC2) were present in the most effective TFs’ list, we have concluded that this complex is one of the major factors in this conversion. Additionally, gene expression profiling of iNSCs, obtained from a different data sets, showed that Sox2 and Ezh2 are two main regulators of the direct reprogramming process. Conclusions Our results provide an insight into molecular events that occur during direct reprogramming of fibroblasts into iNSCs. This information could be useful in simplifying the production of iNSCs, by reducing the number of required factors, for use in regenerative medicine. Electronic supplementary material The online version of this article (doi:10.1186/s13287-015-0045-x) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Moein Yaqubi
- Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
| | - Abdulshakour Mohammadnia
- Department of Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
| | - Hossein Fallahi
- Department of Biology, School of Science, Razi University, Kermanshah, Iran. .,Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.
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Wei C, Li X, Zhang P, Zhang Y, Liu T, Jiang S, Han F, Zhang Y. Characterization of porcine partially reprogrammed iPSCs from adipose-derived stem cells. Reproduction 2015; 149:485-96. [PMID: 25646510 DOI: 10.1530/rep-14-0410] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Partially reprogrammed induced pluripotent stem cells (PiPSCs) have great potential for investigating reprogramming mechanisms and represent an alternative potential material for making genetically modified animals and regenerative medicine. To date, PiPSCs have scarcely been reported in detail when compared with mice and humans. In this study, we obtained PiPSCs from porcine adipose-derived stem cells (pADSCs) by ectopic expression of human transcription factors (OCT4, SOX2, c-MYC, and KLF4) in feeder-free condition. The morphology and proliferation activity of porcine PiPSCs (pPiPSCs) were similar to those of porcine fully reprogrammed iPSCs (pFiPSCs); furthermore, pPiPSCs expressed higher levels of the typical surface molecules (CD29) found in pADSCs. However, pPiPSCs were negative for key proteins (NANOG) connected with stemness and possessed lower differentiation ability in vivo and in vitro. When differentiation-inhibiting factors were withdrawn, pPiPSCs-derived cells (pPiPSC-DCs) showed similar features to pADSCs in many aspects, including proliferation, differentiation, and immunosuppression. When both types of cells were used to produce cloned embryos, we found that the blastocyst formation rate of 19DC (one of the pPiPSC-DC cell lines)-derived cloned embryos was obviously higher than that of others. The total cell number of 19DC-derived blastocysts was significantly higher than the 30DC (one pFiPSC-DC cell line)-derived blastocysts. In all, through limited differentiation ability, the proliferation activity of pPiPSCs is similar to that of pFiPSCs, and pPiPSCs can retain several of the features of pADSCs, which are beneficial to cell therapy. Furthermore, the differentiation of pPiPSCs is more favorable for producing high-quality reconstructed embryos.
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Affiliation(s)
- Chao Wei
- Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and BreedingCollege of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
| | - Xia Li
- Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and BreedingCollege of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
| | - Pengfei Zhang
- Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and BreedingCollege of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
| | - Yu Zhang
- Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and BreedingCollege of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
| | - Tong Liu
- Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and BreedingCollege of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
| | - Shaoshuai Jiang
- Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and BreedingCollege of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
| | - Fei Han
- Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and BreedingCollege of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
| | - Yunhai Zhang
- Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and BreedingCollege of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
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Pandian GN, Sato S, Anandhakumar C, Taniguchi J, Takashima K, Syed J, Han L, Saha A, Bando T, Nagase H, Sugiyama H. Identification of a small molecule that turns ON the pluripotency gene circuitry in human fibroblasts. ACS Chem Biol 2014; 9:2729-36. [PMID: 25366962 DOI: 10.1021/cb500724t] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
A nontransgenic approach to reprogram mouse somatic cells into induced pluripotent stem cells using only small molecules got achieved to propose a potential clinical-friendly cellular reprogramming strategy. Consequently, the screening and identification of small molecules capable of inducing pluripotency genes in human cells are increasingly a focus of research. Because cellular reprogramming is multifactorial in nature, there is a need for versatile small molecules capable of modulating the complicated gene networks associated with pluripotency. We have developed a targeting small molecule called SAHA-PIP comprising the histone deacetylase inhibitor SAHA and the sequence-specific DNA binding pyrrole-imidazole polyamides for modulating distinct gene networks. Here, we report the identification of a SAHA-PIP termed Ì that could trigger genome-wide epigenetic reprogramming and turn ON the typically conserved core pluripotency gene network. Through independent lines of evidence, we report for the first time a synthetic small molecule inducer that target and activate the OCT-3/4 regulated pluripotency genes in human dermal fibroblasts.
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Affiliation(s)
- Ganesh N. Pandian
- Institute
for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Sakyo, Kyoto 606-8502, Japan
| | - Shinsuke Sato
- Institute
for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Sakyo, Kyoto 606-8502, Japan
| | - Chandran Anandhakumar
- Department
of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8501, Japan
| | - Junichi Taniguchi
- Department
of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8501, Japan
| | - Kazuhiro Takashima
- Department
of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8501, Japan
| | - Junetha Syed
- Department
of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8501, Japan
| | - Le Han
- Department
of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8501, Japan
- Shanghai
Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor
Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
| | | | | | - Hiroki Nagase
- Division
of Cancer Genetics, Department of Advanced Medical Science, Nihon University School of Medicine, Tokyo 173-8610, Japan
- Division
of Cancer Genetics, Chiba Cancer Center, Research Institute, 666-2
Nitona-cho, Chuo-ku, Chiba-shi, Chiba 260-8717, Japan
| | - Hiroshi Sugiyama
- Institute
for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Sakyo, Kyoto 606-8502, Japan
- Department
of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8501, Japan
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